Transcript Molecular weight determination

PROTEIN TECHNOLOGY
By
DR ZARINA ZAKARIA
Why to exploit protein
• Information about protein structure has led
to a deeper understanding of the
evolutionary relationships between
species. Exp. To differentiate two species
of Mycobacterium name as M. gastri and
M. kansasii.
• Some inherited disease caused by
alterations in the amino acid sequence of
specific protein. Exp. To detect mutated
protein that caused of inherited
Parkinson's disease.
Techniques in Protein Technology
1.
2.
3.
4.
Isolating
Purification
Chromatography
Electrophoresis
Steps in Protein Purification
1. Protein extraction
- To prepare protein solution
- Total protein only less than 0.1% of total dry
weight.
- Protein are dissolve in buffer solution
- The desired protein is one of many proteins.
1. fractionation
2. chromatography
Steps in Protein Purification
2. Fractionation
- To separate the crude protein.
- Using salting out techniques to precipitate
less soluble protein.
- Remove one-half to two-third of unwanted
protein.
- Also using dialysis technique to separate
high-molecular-weight and LMW.
Steps in Protein Purification
3. Chromatography
- To further fractionate mixture of proteins that
remains after salting-out and dialysis.
• To separate protein mixtures on the basis of
molecular properties such as size, shape, and
weight or certain affinities.
• 3 types of chromatographic methods commonly
used are:
1. gel-filtration chromatography
2. Ion-exchange chromatography
3. Affinity chromatography
i-gel filtration chromatography
• Packed a column with gelatinous polymer
that separates molecules according to
their molecular size.
• Molecules larger than gel pores move
through the column quickly.
• Molecules smaller than gel pores move
thorough the column slowly.
• Differences in movement rate make
available for separate collection.
ii-ion-exchange chromatography
• Separate proteins on the basis of their
charge.
• Packed column with anion-exchange
resins which consist of positively charged
materials.
• Bind protein’s negatively charge groups.
• Remove protein that do not bind to resin
first before recovered the bind proteins.
iii-affinity chromatography
• Uses the unique biologial properties of proteins.
• A special noncovalent binding affinity between
protein and a special molecule (ligand).
• Ligand is covalent bound to insoluble matrix,
which is placed in a column.
• Nonbinding protein molecule will pass through
the column.
• Binding protein removed by altering the
conditions that affect binding.
4-Electrophoresis
• Protein are electrically charge, so they
move in electrically field.
• Molecules separate from each other
because of differences in their net charge.
• Molecules with +ve net charge migrate
toward –vely charge electrode (cathode).
• Molecules with no net charge will not
move at all.
Electrophoresis-continue
• Electrophoresis carry out by using gels
such as polyacrylamide or agarose.
• Similar in function to gel-filtration
chromatography but more effective.
6-Protein characterization
• By knowing the amino acid composition and
amino acid sequencing.
• AA composition is accomplish by determining
the number of each type of amino acid residue
present in the molecule.
• Compose of many process such as:
i-hydrolysis of all peptide bonds with 6N HCL for
10-100 hours.
ii-analysis of resulting amino acid mixture or
hydrolysate by using ion-exchange
chromatography or HPLC.
Protein characterization-continue
• AA sequencing is to determine a protein’s
primary structure.
• Involves several steps:
1-cleavage of all disulfide bonds.
2-determination of the N-terminal and Cterminal amino acids.
3-cleavage of the polypeptide into
fragments.
Protein characterization-continue
4-determination of the sequences of the
peptide fragments.
5-ordering the peptide fragments
Molecular weight determination
• Methods used are:
i-gel-filtration column chromatography
ii-SDS PAGE
iii-ultracentrifugation
Molecular weight determination
• There is correlation between molecular
weight and elution volume, Ve.
• Elution volume is the volume of solvent
required to elute the protein from the
column since it first contacted the gel.
• The MW of the protein is determined by
comparing its relative elution volume (VeVo/Vg) to those several standard
molecules.
Molecular weight determinationcontinue
• Sodium dodecyl sulfate (SDS) is a
powerful negatively charge detergent.
• Polyacrylamide gel electrophoresis
(PAGE) is to separate protein base on
MW.
• Ultracentrifugation uses high centrifugal
forces to determine the protein MW.
X-Ray Crystallography
• To obtain information on three-dimensional
structure about protein.
• Use electromagnetic radiation to resolve
protein structure.
• Crystalline specimen are exposed to X-ray
beam to produce a scattered atoms in the
crystal.
• The scattered image is reconstructed
mathematically to get the structural model.
References
1. McKee, T. & McKee, J.R. Biochemistry:
The molecular Basis of Life.McGraw Hill
2. Horton, Moran, Scrimgeour, perry &
Rawn. Principles of Biochemistry.
Pearson.